The project is a continuation of studies into the immunobiology of murine intestinal intraepithelial lymphocytes (IEL) as a model for understanding human intestinal T cells. The specific aims constitute three interrelated areas of study that will provide an integrated and comprehensive analyses of IEL development, phenotype, and function. Experiments have been designed to identify and characterize precise extrathymic pathway(s) of IEL development from bone marrow stem cells to mature IEL stage. Using methodologies of proven effectiveness, bone marrow-derived IEL precursor cells will be characterized by flow cytometric (FCM) analyses at precise stages during the early phase of IEL reconstitution in euthymic radiation chimeras, and in radiation chimeras constructed from thymectomized mice and congenitally-athymic nude mice. Specific bone marrow stem cells capable of IEL repopulation will be determined. The precise locations of extrathymic development within the small intestine will be identified by immunoperoxidase staining of intestine tissues. Changes in the IEL T cell repertoire will be identified by immunoperoxidase staining of intestine tissues. Changes in the IEL T cell repertoire will be evaluated during development and throughout life. The thymopoietic nature of the murine small intestine will be studied in mice engrafted with sterile intestine, and the possibility of a resident intestinal pluripotent stem cell will be explored. A second series of experiments will characterize adult mouse IEL phenotypically by multi-color FCM analyses using markers not yet applied to IEL studies to correlate IEL developmental lineages with defined IEL subsets. Newly identified and/or novel IEL subsets will be studied by extensive FCM analyses to gain insight into the functional involvement of those cells. Age-related and genetically-determined changes in IEL phenotypes will be studied. IEL-specific monoclonal antibodies recently isolated in this laboratory will be used in studies of IEL, and additional monoclonal antibodies will be generated to identify new IEL- specific markers. CD28 expression will be studied for IEL and other lymphoid tissues, and the molecular heterogeneity of CD28 within the murine immune system will be explored. A third series of experiments will examine functional properties of murine IEL correlated with information from studies of IEL development and phenotype. Proliferation and cytotoxic properties will be studied in phenotypically-defined, Percoll-fractionated IEL subsets and according to states of activation. The functional role of TCR-gammadelta+ IEL expressing Vgamma5 and Vgamma1.2 will be studied using anti-TCR clonotypic antibodies, and antigen recognition by IEL will be explored using recently isolated IEL-derived T cells clones with known reactivities. Collectively, these studies will provide basic information about IEL development, phenotype, and immune function in order to ultimately understand human IEL in health and disease states.